Treatment of adenovical infections with 3&#39;-fluoro-5-halo uracil compounds

ABSTRACT

2&#39;,3&#39;-Dideoxy-3&#39;-fluoro pyrimidine nucleosides particularly 2&#39;, 3&#39;-dideoxy-3&#39;-fluorothymidine have been found to have particularly potent activity against adenovirus infections especially those caused by adenoviruses of scrotype 8. Such compounds are preferably presented in pharmaceutical formulations adapted for ophthalmic administration.

"This is a continuation of copending application(s) Ser. No. 07/504,436filed on Apr. 3, 1990 abandon which is a divisional of 07/234,215 filedAug. 19, 1988, now U.S. Pat. No. 5,070,078".

The present invention relates to certain nucleoside derivatives havingactivity against adenoviruses.

Adenoviruses were first isolated from man in 1953 and at least 41different serotypes representing 6 subgenera have now been identified.Adenoviruses are responsible, for example, for 5% of acute respiratoryinfections in children under 4 years of age and found in 10% ofrespiratory diseases in this age group requiring hospitalisation. Suchconditions are generally associated with pharyngitis, coughing andconjunctivitis. Very often laryngotracheobronchitis occurs whichdevelops into pneumonia in young children, and in fact, 10% of childhoodpneumonias are due to adenovirus infection and are often fatal inchildren under 2 years of age.

In the older population adenoviruses are often responsible forpharyngoconjunctival fever and acute respiratory disease ininstitutionalized persons where it has been known to have a fataloutcome. The viruses are often associated with pertussis syndrome,haemorrhagic cystitis, meningitis, diarrhoea and epidemickeratoconjunctivitis. The latter condition is characterised by rapidconjunctival involvement with pain, photophobia, lymphadenopathy andsubsequent keratitis. The syndrome may last for several weeks but thecorneal opacity may endure for several years. The patient is thereforedisabled to varying degrees over a period of time. It has been shownthat adenovirus type 8 is the major etiologic agent in this particularaspect of adenovirus disease. Adenovirus disease is particularly severein children with severe combined immunodeficiency disease (SCID) and inimmunocompromised hosts. Adenoviruses are recognised also asincreasingly more common in patients with Acquired Immune DeficiencySyndrome (AIDS) and in bone marrow transplant recipients. There hashitherto been no useful antiviral compound that is effective in thetreatment of adenoviral infections and there is also no adequatevaccine.

We have now discovered that compounds of formula (I) below have potentactivity against adenoviruses particularly those of type 8.

The present invention accordingly provides compounds of formula (I)##STR1## (wherein R represents a hydrogen atom, C₁₋₄ alkyl (e.g.methyl), or a halogen atom (e.g. a chlorine, bromine or iodine atom) andphysiologically acceptable salts and esters thereof, for use in thetreatment of an adenovirus infection. Such compounds, salts and estersare hereafter referred to as compounds according to the invention.

Particularly preferred compounds of formula (I) by virtue of theirespecially potent activity against adenoviruses are:

1. 2',3'-dideoxy-3'-fluorothymidine

2',3'-dideoxy-3'-fluorouridine

3. 5-bromo-1-(2,3-dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)uracil

4. 5-iodo-1-(2,3-dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)uracil

5. 5-chloro-1-(2,3-dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)uracil.

Compound No 1 referred to above has particularly high anti-adenovirusactivity especially against serotypes 5 and 8.

In a further aspect of the present invention, there is provided the useof a compound according to the invention in the manufacture of amedicament for the treatment of adenovirus infections.

The present invention further provides a method for the treatment of anadenovirus infection in a human subject which comprises administering tothe said human subject an effective amount of a compound according tothe invention.

Examples of adenovirus infections which may be treated in accordancewith the present invention include the clinical conditions referred toabove, and particularly adenoviral infections of the eye.

Preferred esters of the compounds of formula (I) include carboxylic acidesters in which the non-carbonyl moiety of the ester grouping isselected from straight or branched chain alkyl, alkoxyalkyl (e.g.methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g.phenoxymethyl), aryl (e.g. phenyl optionally substituted by halogen,C₁₋₄ alkyl or C₁₋₄ alkoxy); sulphonate esters such as alkyl- oraralkylsulphonyl (e.g. methanesulphonyl); and mono-, di- ortri-phosphate esters. With regard to the above-described esters, unlessotherwise specified, any alkyl moieties present in such estersadvantageously contain 1 to 18 carbon atoms, particularly 1 to 4 carbonatoms. Any aryl moiety present in such esters advantageously comprises aphenyl group. Any reference to any of the above compounds also includesa reference to a pharmaceutically acceptable salt thereof.

Examples of pharmaceutically acceptable salts of the compound of formula(I) and its pharmaceutically acceptable derivatives include base salts,e.g. derived from an appropriate base, such as alkali metal (e.g.sodium), alkaline earth metal (e.g. magnesium) salts, ammonium and NX₄ ⁺(wherein X is C₁₋₄ alkyl).

Certain of the compounds of formula (I) above have been previouslydescribed in the literature. For example, the compound of formula (I)above in which R represents a hydrogen atom is described by G. Kowolliket al., Journal f. prakt Chemie, 315(5), 1973, 895-900; and the compoundof formula (I) in which R represents a methyl group is described in UKPatent Specification No. 1189973. Also the compound of formula (I) inwhich R represents a bromine atom is referred to in European PatentSpecification No. 254268.

The above compounds of formula (I) in which R represents a chlorine oriodine atom and their physiologically acceptable salts and esters are,however, new compounds and represent further features of the presentinvention.

The compounds according to the invention may be administered to humansfor the treatment of adenovirus infections and eye infections inparticular, by any suitable route including oral, rectal, nasal, topical(including buccal and sublingual), vaginal and parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal). It will beappreciated that the preferred route will vary with the condition andage of the recipient, the nature of the infection and the chosen activeingredient.

In general a suitable dose will be in the range of 3.0 to 120 mg perkilogram body weight of the patient per day, preferably in the range of6 to 90 mg per kilogram body weight per day and most preferably in therange 15 to 60 mg per kilogram body weight per day. The desired dose ispreferably presented as two, three, four, five, six or more sub-dosesadministered at appropriate intervals throughout the day. Thesesub-doses may be administered in unit dosage forms, for example,containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably50 to 700 mg of the compound per unit dosage form.

While it is possible for the compounds according to the invention to beadministered alone it is preferably to present it as a pharmaceuticalformulation. The formulations of the present invention comprise at leastone compound according to the invention together with one or moreacceptable carriers thereof and optionally other therapeutic agents.Each carrier must be "acceptable" in the sense of being compatible withthe other ingredients of the formulation and not injurious to thepatient. Formulations include those suitable for oral, rectal, nasal,topical (including buccal and sublingual), ophthalmic, vaginal orparenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. Such methods include the step of bringinginto association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then if necessary shaping the product.

In view of the activity of the compounds according to the inventionagainst adenovirus infection of the eye, as referred to above, it isparticularly preferred to present the compounds according to theinvention in the formulations adapted for ophthalmic administration.

Such formulations for opthalmic administration include eye drops andophthalmic ointments, creams, suspensions and lotions.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or any other suitable preservative. The resulting solution may thenbe clarified by filtration, transferred to a suitable container which isthen sealed and sterilised by autoclaving. Alternatively, the solutionmay be sterilised by filtration and transferred to the container by anaseptic technique. Examples of bactericidal and fungicidal agentssuitable for inclusion in the drops are phenylmercuric nitrate oracetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidineacetate (0.01%).

Lotions according to the present invention include sterile aqueoussolutions optionally containing a preservative and may be prepared bymethods similar to those for the preparation of drops.

Creams or ointments according to the present invention are semi-solidformulations of the active ingredient particularly for opthalmicapplication. They may be made by mixing the active ingredient infinely-divided or powdered form alone or in solution or suspension in anaqueous or non-aqueous fluid, with a greasy or non-greasy basis. Thebasis may comprise hydrocarbons such as hard, soft or liquid paraffin,glycerol, beeswax, a metallic soap; a mucilage, an oil of natural originsuch as almond, corn, arachis, castor or olive oil, wool fat or itsderivatives, or a fatty acid such as stearic or oleic acid together withan alcohol such as propylene glycol or macrogols. The formulation mayincorporate any suitable surface active agent such as sorbitan esters orpolyoxyethylene derivative thereof. Suspending agents such as naturalgums, cellulose derivatives or inorganic materials such as silicaceoussilicas; and other ingredients such as lanolin may also be included.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus or paste. Oral formulations may further includeother agents conventional in the art, such as sweeteners, flavouringagents and thickeners.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally incorporatinga binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, disintegrant (e.g. sodium starch glycollate,cross-linked povidone, cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Moulded tablets may be made bymoulding in a suitable machine a mixture of the powdered compoundsmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be present aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose ordaily sub-dose, as herein above recited, or an appropriate fraction, ofthe compound according to the invention.

The compounds according to the invention may be prepared in conventionalmanner. Thus, for example, compounds of formula (I) particularly thecompound wherein R represents a methyl group may be prepared asdescribed in UK Patent Specification No. 1189973. The compound offormula (I) wherein R represents a hydrogen atom may be prepared asdescribed for example by G. Kowollik et al, J. Prakt. Chem. 1973,315(5), 895-900.

The compounds of formula (I) in which R represents a halogen(particularly a chlorine, bromine or iodine) atom may be prepared forexample by halogenating a corresponding compound of formula (I) in whichR represents a hydrogen atom and in which the 5'-hydroxy group isblocked, for example by an acyl group such a p-toluoyl group.

Halogenation of the above starting material may be effected inconventional manner, for example iodination using iodine monochloride,e.g. in methylene dichloride, bromination using bromine e.g. in glacialacetic acid and chlorination using a chlorine complex of iodobenzene,e.g. in glacial acetic acid.

The above toluoyl derivative may be prepared by treating the appropriatecompound of formula (I) with for example p-toluoyl chloride, e.g. inpyridine. After halogenation as described above, the p-toluoylprotecting group may be removed for example by sodium methoxide inmethanol.

The parent compound of formula (I) may be converted into aphysiologically acceptable ester by reaction with an appropriateesterifying agent, e.g. an acid halide or anhydride. The compound offormula (I), including esters thereof, may be converted intopharmaceutically acceptable salt thereof in conventional manner, e.g. bytreatment with an appropriate base. An ester or salt may be convertedinto the parent compound, e.g. by hydrolysis.

The following Examples are intended for illustration only and are notintended to limit the scope of the invention in any way. The term`active ingredient` as used in the Examples means a compound of formula(I) or a physiologically acceptable salt or ester thereof.

Example 11-(2,3-Dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)-5-iodouracil

p-Toluoyl chloride (freshly distilled, 325 mg, 2.10 mmol) was added to asolution of 1-(2,3-dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)uracil(440 mg, 1.91 mmol) in dry pyridine (10 ml). The solution was stirred at50° for 1.5 hour, and then at 25° for 18 hours. The pyridine wasevaporated and the residue dissolved in CHCl₃ (25 ml). This solution wasextracted with 1M H₂ SO₄ (5 ml), then H₂ O (2×10 ml), and dried (MgSO₄).Evaporation of CHCl₃ left a colourless glass (0.72 g) which waschromatographed on silica gel. Elution with 2% MeOH-CHCl₃ gave the5'-O-toluoyl derivative as white solid foam (0.66 g, 90%);chromatographically homogeneous on TLC plates (silica gel developed with5% MeOH-CHCl₃); structure confirmed by 'H-NMR.

The 5'-O-toluoyl derivative of1-(2,3-dideoxy-3-fluoro-β-D-erythropentofuranosyl)uracil (200 mg, 0.574mmol), iodine monochloride (139 mg, 0.861 mequiv), and CH₂ Cl₂ (10 ml)were refluxed for 2 hours. The solution was decolourised with a minimumof 2% aqueous NaHSO₃ (ca. 2 ml). The aqueous layer was separated and theCH₂ Cl₂ layer washed with H₂ O (2×5 ml) and dried (MgSO₄). Evaporationof CH₂ Cl₂ left a cream colored solid foam (0.25 g) which was dissolvedin MeOH (10 ml) and stirred with sodium methoxide (0.57 mmol) under N₂at 25° for 18 hours. The solution was neutralized with Dowex 50W-X8 (H⁺form) resin. The resin was filtered off, washed with MeOH, and thecontents of the methanol filtrate wash chromatographed on silica gel.Elution with 10% MeOH-CH₂ Cl₂ gave a product as a white solid (0.135 g).Recrystallization from EtOH gave title compound as white crystals (115mg, 55% overall yield); m.p. 197.5°-198° dec; Anal: Calcd for C₉ H₁₀ FN₂O₄ : C, 30.36; H, 2.83; N, 7.87; F, 5.34; I, 35.64. Found: C, 30.50; H,2.85; N, 7.85; F, 5.31; I, 35.51; UV λ max (H₂ O): 286 nM; structurefurther confirmed by 'H-NMR and mass spectrum.

EXAMPLE 25-Bromo-1-(2,3-dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)uracil

The 5'-O-toluoyl derivative of1-(2,3-dideoxy-3-fluoro-β-D-erythropentofuranosyl)uracil, prepared asdescribed in Example 1, (200 mg, 0.574 mmol), glacial acetic acid (5 ml)and bromine (0.08 ml of an 8M solution in glacial acetic acid, 0.64mequiv.) were stirred at 25° for 18 hours. The solution was evaporatedto a glass which was treated with sodium methoxide and methanol asdescribed in Example 1. Product was eluted from a silica gel column with5% MeOH-CH₂ Cl₂ as white crystals (135 mg). Recrystallization from EtOHgave title compound as white crystals (115 mg, 64% overall yield); m.p.193.5°-194° dec.; Anal Calcd for C₉ H₁₀ BrFN₂ O₄ : C, 34.97; H, 3.26; N,9.06; Br, 25.85; F, 6.15. Found: C, 35.07; H, 3.31; N, 9.00; Br, 25.78;F, 6.04; UV λ max (H₂ O): 277 nM; structure further confirmed by 'H-NMRand mass spectrum.

EXAMPLE 35-Chloro-1-(2,3-dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)uracil

The chlorine complex of iodobenzene was freshly prepared as described inthe literature (see M. J. Robins, et al., Can. J. Chem. 1982, 60, 554)and 246 mg (0.895 mmol) added to a solution of the 5'-O-toluoylderivative of 1-(2,3-dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)uracil(260 mg, 0.746 mmol), prepared as described in Example 1, in glacialacetic acid (4 ml). This solution was maintained at 80° under nitrogenfor 20 minutes and evaporated to a white solid foam. Treatment withsodium methoxide in methanol was carried out as in Example 1.Chromatography on 2 mm thick silica gel plates (20×20 cm) developed inCHCl₃ : MeOH: NH₄ OH/180:20:1 was required to separate 5-chlorouracil(slightly greater R_(f)) from title compound, isolated as an off-whitesolid (46 mg). Trituration in methanol gave title compound as whiteneedles (34 mg); m.p. 183°-184°; Anal. Calcd for C₉ H₁₀ ClFN₂ O₄ : C,40.85; H, 3.81; Cl, 13.40; N, 10.59. Found: C, 40.71; H, 3.85; Cl,13.31; N, 10.55; UV λ max (H₂ O) 275 nM; structure further confirmed by'H-NMR and mass spectrum.

EXAMPLE 4: Tablet Formulations

The following formulations A, B and C are prepared by wet granulation ofthe ingredients with a solution of povidone, followed by addition ofmagnesium stearate and compression.

    ______________________________________                                        Formulation A                                                                                  mg/tablet                                                                              mg/tablet                                           ______________________________________                                        (a)  Active ingredient 250        250                                         (b)  Lactose B.P.      210        26                                          (c)  Povidone B.P.     15         9                                           (d)  Sodium Starch Glycollate                                                                        20         20                                          (e)  Magnesium Stearate                                                                              5          3                                                                  500        300                                         ______________________________________                                    

    ______________________________________                                        Formulation B                                                                                  mg/tablet                                                                              mg/tablet                                           ______________________________________                                        (a)  Active ingredient 250        250                                         (b)  Lactose           150        --                                          (c)  Avical PH 101     60         26                                          (d)  Povidone B.P.     15         9                                           (e)  Sodium Starch Glycollate                                                                        20         12                                          (f)  Magnesium Stearate                                                                              5          3                                                                  500        300                                         ______________________________________                                    

    ______________________________________                                        Formulation C                                                                                mg/tablet                                                      ______________________________________                                        Active Ingredient                                                                              100                                                          Lactose          200                                                          Starch           50                                                           Povidone B.P.    5                                                            Magnesium Stearate                                                                             4                                                                             359                                                          ______________________________________                                    

The following formulations, D and E, are prepared by direct compressionof the admixed ingredients. The lactose used in formulation E is of thedirect compression type.

    ______________________________________                                        Formulation D                                                                                   mg/capsule                                                  ______________________________________                                        Active Ingredient   250                                                       Pregelatinised Starch NF15                                                                        150                                                                           400                                                       ______________________________________                                    

    ______________________________________                                        Formulation E                                                                                mg/capsule                                                     ______________________________________                                        Active Ingredient                                                                              250                                                          Lactose B.P.     150                                                          Avicel           100                                                          Magnesium Stearate                                                                              5                                                                            505                                                          ______________________________________                                    

Formulation F (Controlled Release Formulation)

The formulation is prepared by wet granulation of the ingredients(below) with a solution of povidone followed by the addition ofmagnesium stearate and compression.

    ______________________________________                                                            mg/tablet                                                 ______________________________________                                        (a)    Active Ingredient  500                                                 (b)    Hydroxypropylmethylcellulose                                                                     112                                                        (Methocel K4M Premium)                                                 (c)    Lactose B.P.       35                                                  (d)    Povidone B.P.C.    28                                                  (e)    Magnesium Stearate 7                                                                             700                                                 ______________________________________                                    

EXAMPLE 5: Capsule Formulations

Formulation A

A capsule formulation is prepared by admixing the ingredients ofFormulation D in Example 1 above and filling into a two-part hardgelatin capsule. Formulation B (infra) is prepared in a similar manner.

    ______________________________________                                        Formulation B                                                                                   mg/capsules                                                 ______________________________________                                        (a)    Active Ingredient                                                                              250                                                   (b)    Lactose B.P.     143                                                   (c)    Sodium Starch Glycollate                                                                       25                                                    (d)    Magnesium Stearate                                                                             2                                                                             420                                                   ______________________________________                                    

    ______________________________________                                        Formulation C                                                                                 mg/capsule                                                    ______________________________________                                        (a)      Active Ingredient                                                                           250                                                    (b)      Macrogol 4000 BP                                                                            600                                                                           600                                                    ______________________________________                                    

Capsules are prepared by melting the macrogol 4000 BP, dispersing theactive ingredient in the melt and filling the melt into a two-part hardgelatin capsule.

    ______________________________________                                        Formulation D                                                                               mg/capsule                                                      ______________________________________                                        Active Ingredient                                                                             250                                                           Lecithin        100                                                           Arachis Oil     100                                                                           450                                                           ______________________________________                                    

Capsules are prepared by dispersing the active ingredient in thelecithin and arachis oil and filling the dispersion into soft, elasticgelatin capsules.

Formulation E (Controlled Release Capsule

The following controlled relase capsule formulation was prepared byextruding ingredients (a), (b) and (c) using an extruder, followed byspheronisation of the extrudate and drying. The dried pellets were thencoated with release-controlling membrane (d) and filled into atwo-piece, hard gelatin capsule.

    ______________________________________                                                          mg/capsule                                                  ______________________________________                                        (a)    Active Ingredient                                                                              250                                                   (b)    Microcrystalline Cellulose                                                                     125                                                   (c)    Lactose BP       125                                                   (d)    Ethyl Cellulose   13                                                                           513                                                   ______________________________________                                    

EXAMPLE 6: Injectable Formulation

    ______________________________________                                        Formulation A.                                                                ______________________________________                                        Active Ingredient              0.200 g                                        Hydrochloric acid solution, 0.1M                                                                  q.s. to pH 4.0 to 7.0                                     Sodium hydroxide solution, 0.1M                                                                   q.s. to pH 4.0 to 7.0                                     Sterile water       q.s. to    10 ml                                          ______________________________________                                    

The active ingredient is dissolved in most of the water (35°-40° C.) andthe pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or thesodium hydroxide as appropriate. The batch is then made up to volumewith the water and filtered through a sterile micropore filter into asterile 10 ml amber glass vial (type 1) and sealed with sterile closuresand overseals.

    ______________________________________                                        Formulation B.                                                                ______________________________________                                        Active Ingredient         0.125   g                                           Sterile, pyrogen-free, pH 7 citrate buffer, q.s. to                                                     25      ml                                          ______________________________________                                    

EXAMPLE 7: Intramuscular Injection

    ______________________________________                                        Active Ingredient       0.20   g                                              Benzyl Alcohol          0.10   g                                              Glycofurol 75           1.45   g                                              Water for Injection q.s. to                                                                           3.00   ml                                             ______________________________________                                    

The active ingredient is dissolved in the glucofurol. The benzyl alcoholis then added and dissolved, and water added to 3 ml. The mixture wasthen filtered through a sterile micropore filter and sealed in sterile 3ml amber glass vials (type 1).

EXAMPLE 8

    ______________________________________                                        Eye Drops                                                                     ______________________________________                                        Active Ingredient        0.20   g                                             Benzalkonium Chloride    0.01   g                                             Isotonic, phosphate buffer, pH 7 to                                                                    10.00  ml                                            ______________________________________                                    

The active ingredient is dissolved in most of the Isotonic phosphatebuffer. The benzalkonium chloride is added and dissolved and the batchmade to volume with further Isotonic phosphate buffer. The solution isfiltered through a sterile, sterilising grade filter and packed intosterile eye drop bottles and sealed.

EXAMPLE 9

    ______________________________________                                        Eye Ointment                                                                  ______________________________________                                        Active Ingredient        0.5   g                                              Sterile White Soft Paraffin to                                                                         5.0   g                                              ______________________________________                                    

The active ingredient is sterilised by dry heat and mixed with theSterile White Soft Paraffin under aseptic conditions. The resultingdispersion is packed into sterile ophthalmic ointment tubes.

EXAMPLE 10

    ______________________________________                                        Ophthalmic Suspension                                                         ______________________________________                                        Active Ingredient      0.1    g                                               Polysorbate 80         0.005  g                                               Thiomersal             0.001  g                                               Propylene Glycol       0.1    g                                               Water for Injection to 5.0    ml                                              ______________________________________                                    

The Polysorbate 80, Thiomersal and Propylene Glycol are dissolved inmost of the Water for Injections.

The solution is sterilised by filtration. The sterile, micronised activeingredient is dispersed in the solution under aseptic conditions. Thebatch is made up to volume with further Water for Injections which hasbeen sterilised previously. The product is packed into sterile eye dropbottles and sealed.

Antiviral Activity

The anti-adenoviral activity of Compound No. 1 referred to above wastested as follows:

Cells of human epithelial cell line NCTC 2544 (EP) were seriallypassaged from 75 cm² tissue culture flasks (Corning) at a split ratio of1/20 every 7 days. The growth medium was Eagles BHK medium supplementedwith 10% foetal bovine serum and contained 2.5% of 4.4% sodiumbicarbonate solution together with 1% of penicillin (5000 IU ml⁻¹) andstreptomycin (600 μg ml⁻¹) (Flow).

A stock of adenovirus of type 8 was prepared in 25 cm² tissue cultureflasks. Flasks were seeded with 2×10⁶ EP cells in growth medium andincubated overnight at 37° C. The medium was decanted and 1.0 ml ofstock virus suspension was added to the flask. After 1.0 hr adsorption10 ml of maintenance medium (as above but with 2% serum) was added tothe flask.

The flask were incubated at 37° C. After 72 hrs the classical `bunch ofgrapes` cytopathic effect was visible. The flasks were then frozen andthawed 3 times to liberate the virus and stored at -70° C. Viral titreswere usually of the order of 4×10⁸ pfu ml⁻¹.

The plaque assay was performed in 50 mm diameter plastic tissue culturedishes which were initially seeded with 2×10⁶ EP cells in growth mediumand incubated overnight in 5% CO₂ at 37° C. Adenovirus was inoculated onto the cell monolayers in 1.0 ml serum-free medium and allowed to adsorbat 37° C. in a CO₂ incubator for 1 hr. After adsorption excess inoculumwas removed and 9.0 ml overlay medium was added. This consisted of 0.5%agarose, Eagles BHK medium, 2.5% of 4.4% sodium bicarbonate solution,1.5% foetal bovine serum, 1% of penicillin/streptomycin solution and0.5% of 3M magnesium chloride solution. From day 5 when plaques could beseen in the unstained culture, the cultures were fixed in 10% of 40%formaldehyde solution in phosphate buffered salt solution. After onehour the overlay gel was rinsed from the plate with tap water and theexposed monolayer stained with 2 ml of 0.5% methyl violet in 5%methanol.

EP cells allowed the productive growth of adenovirus and plaques wereclearly visible.

The following results was obtained for the activity of the aboveCompound against adenovirus of serotype 8:

    ______________________________________                                        Compound No.    IC.sub.50 (μM)                                             ______________________________________                                        1               0.005                                                         ______________________________________                                    

Cytotoxicity Determination

Cytotoxicity was determined by means of the trypan blue exclusionmethod. NCTC 2544 cells were prepared in growth medium and aliquotsadded to 25 cm³ tissue culture flasks at approximately 1×10⁵ cells/ml.At 24 hours the flasks were divided and replenished with growth medium.One half of the flasks received growth medium containing 10 folddilutions of compound thus exposing the cells to 1, 10, and 100 μMconcentrations. At 24, 48, and 72 hours representative flasks wereremoved from the incubator and the monolayers washed to remove debris.The cells were resuspended in growth medium and aliquots mixed with anequal volume of trypan blue. Stained and unstained cells were counted ina haemocytometer. Cell counts were adjusted to cells/ml. An estimate ofthe growth and viability of treated and untreated cells was then made.

The 50% cell culture inhibitory concentration of compound No. 1) was 38μM at 72 hours.

Preparation of1-(2-Deoxy-5-(p-chlorobenzoyl)-β-D-threo-pentofuranosyl)thymine

1-(2-Deoxy-β-D-threo-pentofuranosyl)thymine (J. J. Fox and N. C. MillerJ. Org. Chem., 1963, 28, 936) (49.0 g) was dissolved in 500 ml ofanhydrous pyridine in a 1 L round bottom flask equipped with an additionfunnel, nitrogen inlet and mechanical stirrer. The solution was cooledto 5° C. in an ice bath and 4-chlorobenzoyl chloride (35.4 g, 0.202 mol)was added dropwise over 30 minutes. The solution was allowed togradually warm to ambient temperature. The reaction was quenched withwater (20 ml), most of the pyridine removed by rotary evaporation andthe residual oil dissolved in ethyl acetate (1 L). The organic solutionwas washed with 700 ml portions of water, 1.2N HCl and 10% aqueoussodium bicarbonate and dried over sodium sulphate. The mixture was thenfiltered and solvent removed by rotary evaporation affording1-(2-deoxy-5-(p-chlorobenzoyl)-D-threopentofuranosyl)thymine (62.1 g,80.7%) as an off-white solid suitable for use in the next step: 1H NMR(DMSO-d6) 1.76 (d, J=0.9 Hz, 3H, C-5 CH₂), 2.1-2.6 (m, 2H, C-2'),3.8-4.5 (m, 4H, C-3'H, C-4'H and C-5'H), 5.55 (d, J=2.6 Hz, 1H, C-3'OH),6.13 (doublet of doublets, J=2.8 Hz and 8.3 Hz, 1H, C-1'H), 7.58 (d,J=8.6 Hz, 2H, ArH), 7.81 (m, 1H, C-6H), 7.85 (d, J=8.6 Hz, 2H, ArH);TLC, Rf=0.72, silica gel, CHCl₃ :CH₃ OH/90:10.

Preparation of 3'-Deoxy-3'-fluoro-thymidine

A suspension of1-(2-deoxy-5-(p-chlorobenzoyl)-β-D-threo-pentofuranosyl)thymine (61.0 g)and potassium carbonate (30.0 g) in methylene chloride (700 ml) wascooled to -78° C. in a 2 L round bottom flask equipped with a mechanicalstirrer, addition funnel and nitrogen inlet. Diethylaminosulfurtrifluoride (30 ml) was added dropwise over 10 minutes andstirring was continued an additional 2.5 h at -78° C. The solution waswarmed to room temperature, transferred to a separatory funnel andwashed with ice water (700 ml), and 500 ml portions of water, 1.2Nhydrochloric acid and 10% aqueous sodium bicarbonate. The solvent wasremoved by rotary evaporation and the residue was dissolved in methanol(500 ml). Solid sodium bicarbonate (15 g) was added and the suspensionwas heated 2 h at reflux. The mixture was cooled to room temperature,filtered, solvent removed by rotary evaporation and the residual oilpurified by flash chromatography using a 3×36 inch volume of flashsilica gel. The column was eluted with 96:4/CHCl₃ :CH₃ OH whilecollecting 200 ml fractions. Fractions 29-41 were concentrated by rotaryevaporation, the residue slurried in acetone (100 ml) and the solidscollected by filtration affording 7.70 g (19.9%) of3'-deoxy-3'-fluorothymidine: mp=169°-171° C.; TLC, Rf=0.53, Silica Gel,90:10/CHCl₃ :CH₃ OH; Elemental Analysis Calc C, 49.18; H, 5.37; N,11.47; Found C, 49.00; 5.38; N, 11.40.

We claim:
 1. The method of treating an adenovirus serotype 8 infectionin a human subject comprising administering to said subject having saidinfection an effective adenovirus serotype 8 infection treatment amountof 1-(2,3-dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)-5-iodouracil or apharmaceutically acceptable salt thereof.
 2. The method of treating anadenovirus serotype 8 infection in a human subject comprisingadministering to said subject having said infection an effectiveadenovirus serotype 8 infection treatment amount of the compound5-chloro-1-(2,3-dideoxy-3-fluoro-β-D-erythropentofuranosyl) uracil or apharmaceutically acceptable salt thereof.